title: The RPA32 subunit of human replication protein A contains a single-stranded DNA-binding domain.
abstract: Replication protein A (RPA) is a conserved nuclear single-stranded DNA (ssDNA)-binding protein. Human RPA (hRPA) comprises three subunits of approximately 70, 32, and 14 kDa (hRPA70, hRPA32 and hRPA14). RPA is known to bind ssDNA through two ssDNA-binding domains in the RPA70 subunit. Here, we demonstrate that the complex of hRPA32 and hRPA14 has an ssDNA-binding domain. Limited proteolysis of the hRPA14.32 complex defined a core dimer composed of the central region of hRPA32 (amino acids 43-171) and RPA14. The core dimer bound ssDNA with an affinity of approximately 10-50 microM, which is at least 100-fold more avid than the DNA-binding affinity of the intact dimer. Analysis of the predicted secondary structure of hRPA32 suggests that amino acids 63-150 of hRPA32 form an ssDNA-binding domain similar in structure to each of those in hRPA70. The complex of hRPA14 and hRPA32-(43-171) in turn formed a trimeric complex with the C-terminal region of hRPA70 (amino acids 436-616). The ssDNA-binding affinity of this trimeric complex was 3 to 5-fold higher than hRPA14.32-(43-171) alone, suggesting a role for the C terminus of hRPA70 in ssDNA binding.
authors.affiliation: Ontario Cancer Institute, Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 2M9, Canada.
authors.forename: E
authors.initials: E
authors.lastname: Bochkareva
authors.forename: L
authors.initials: L
authors.lastname: Frappier
authors.forename: A M
authors.initials: AM
authors.lastname: Edwards
authors.forename: A
authors.initials: A
authors.lastname: Bochkarev
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000032:analysis / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D051716:Replication Protein A; D016415:Sequence Alignment; D017421:Sequence Analysis; D012697:Serine Endopeptidases / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
chemical_list: D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D010446:Peptide Fragments; C497167:RPA1 protein, human; D011994:Recombinant Proteins; D051716:Replication Protein A; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin